Crystallization of streptomycins



Patented Mar. 31, 1953 CRYSTALLIZATION F STREPTOMYCINS Leon J. Heuser,Robbinsville, N. J., assignor to Mathieson Chemical Corporation, NewYork, N. Y., a corporation of Virginia No Drawing. Application September23, 1950, Serial No. 186,487

8 Claims.

This invention relates to the crystallization of streptomycins.

[The term streptomycins, or a streptomycin identifies the genus (or amember of the genus) consisting of streptomycin, mannosidostreptomycin,and streptomycin complex.l

Streptomycins have been purified by various methods, the early methodsbeing generally complicated and/or inefficient (and consequentlyexpensive). Recently, simple, efiicient and otherwise advantageousmethods of purifying streptomycins have been provided, these improvedmethods being the subject of certain U. S. patent applications [Lott,Bernstein and Heuser applications Serial Numbers 767,851 and 767,852,filed August 9, 1947, now Patents Nos. 2,537,933 and 2,537,934,respectively, dated January 9, 1951]. These improved purificationmethods are based on the findings that: the streptomycins interact withsurface-active agents of the organicallysubstitutedpolybasic-inorganic-acid type to form salt-type combinations which aremuch less soluble in water (and more soluble in certain organicsolvents) than the streptomycins; and that such salt-type derivatives ofthe streptomycins may be recovered and converted into simplemineral-acid salts of streptomycins in purified form.

One of these methods (of. Patent No. 2,537,933) essentially comprisesintimately contacting an aqueous solution of the impure streptomycin e.g., a primary streptomycin-containing liquid, or an aqueous solution ofa partially-purified streptomycin, such as the eluate from the charcoaladsorbate) with a surface-active agent of the organically-substitutedpolybasic-inorganicacid type and a substantially water-immiscibleorganic solvent for soaps (including invert soaps) preferably with asubstantially water-immiscible aliphatic alcohol; separating the organicsolvent phase; and recovering the salt-type derivative of thestreptomycin therein and/or converting it to awater-soluble salt of thestreptomycin (as by intimately contacting the organic solvent solutionwith an aqueous, water-soluble, relatively-strong acid, andrecoveringthe aqueous phase).

The other of these methods (of. Patent No. 2,537,934) essentiallycomprises: treating the impure streptomycin with the surface-activeagent in a solvent for the reactants (especially water) recovering theformed, relatively water-insoluble salt-type combination derived fromthe streptomycin and surface-active agent (by filtration, for example);and converting the latter combination into a water-soluble salt of thestreptomycin (as by dissolving the combination in a solvent therefor,treating the solution with an aqueous, watersoluble relatively-strongacid, and recovering the formed water-soluble salt of the streptomycin).

The water-soluble salt (of the streptomycin) obtained by such improvedpurification treatment is considerably purer than the streptomycintreated, and the recovery of streptomycin activity is of a high order(it being possible to obtain uniformly high yields of relatively-purestreptomycin complex, for example, having a potency above about 400units/mg).

It is the object of. this invention to provide certain crystallinestreptomycins, and advantageous methods of obtaining them.

The crystalline streptomycins of this invention are the sesquisulfatesand sesquiphosphates, these crystalline salts being of a particulartautomeric form, as explained hereinafter. [These crystallinestreptomycins may be obtained by treatment of certain of theaforementioned salt-type combinations of streptomycins andsurface-active agents with sulfuric acid and phosphoric acid undercertain conditions, but the yields are unsatisfactory, and the productsare often objectionably colored] The advantageous methods of thisinvention essentially comprise: mixing (A) an organic-solvent solutionof a salt-type combination derived from the streptomycin and a member ofthe group consisting of C4H9CH (Cal-I5) C2H4CH(SO4Na) CsiHiCH (CzHs) 2and [preferably the former], the organic solvent being substantiallywater-immiscible and being'a nonsolvent for the desired salt ofstreptomycin, and (B) an aqueous solution of a weak-base salt of an acidof the group consisting of sulfuric and phosphoric; and recovering theformed crystalline salt of the streptomycin. Crystalline sesquisulfatesand sesquiphosphates of the streptomycins, especially streptomycinsesquisulfate, are thus obtained in high yield.

By virtue of their high purity and low coloration, the crystallinestreptomycins obtained in the practice of this invention are especiallysuitable for conversion to the corresponding dihydrostreptomycins bycatalytic hydrogenation or electrolytic reduction. Moreover, thesecrystalline streptomycins may be converted to the dihydrostreptomycinswhile in the form of a crystal slurry.

The crystalline compounds of this invention differ from thecorresponding amorphous compounds in a respect other than purity orcrystalline form. Thus, there are indications that they are alsodifferent tautomeric forms of the amorphous compounds. These indicationsinclude the facts that: crystalline streptomycin sesquisulfate, forexample, is difiicultly soluble in water at its own pH, whereas theamorphous compound is readily soluble at that pH; pure amorphousstreptomycin sesquisulfate cannot be crystallized by conventionalprocedures; aging of the tergitates enhances the yield of thecrystalline compound; and crystalline streptomycin sesquisulfate may beobtained from crystalline streptomycin hydrochloride, but not from-pureamorphous streptomycin hydrochloride, by treatment with sulfuric acid oruanidine sulfate.

The following examples are illustrative of the invention:

Example 1 20 g. substantially-dry streptomycin tergitate [containingabout water, obtained from streptomycin and C4H9CH (C2115)C2I-I4CH(SO4Na) C2H4CH(C2H5) 2 (Tergitol Penetrant '7) as described inapplication Serial No. 767,852, referred to hereinbeforel, or acorresponding amount of the wet tergitate, is dissolved in 100 ml.n-butanol. The solution is filtered, and the filtrate washed with 100ml. 0.3% sulfuric acid; and the butanol phase is recovered and added to100 ml. of a 5% aqueous solution of guanidine sulfate at such rate as toprevent any precipitation (over an about fiveminute period) whileagitating, the guanidine sulfate solution having priorly been adjustedto pH 3.2 by addition of dilute sulfuric acid and heated to 65-70 C.[Heating is not essential for crystallization; but without heating,smaller crystals are formed] The reaction mixture is then allowed tocool to C, over about a 1 /2 hour period, during which time streptomycinsesquisulfate precipitates from the aqueous phase as crystals of thecoarseness of table salt. The crystals are filtered on, and dried undervacuum (yield about Assays: chemical-about 800 units/mg; bio-assayabout833 units/mg. [The coarseness of the crystals enables them to be 1filtered and worked with case on a perforated basket centrifuge] Thestreptomycin sesquisulfate thus obtained is insoluble in methanol, anddifilcultly soluble in water at its own pH (in contrast with amorphousstreptomycin sesquisulfate), but gradually dissolves at that pH andreadily dissolves at about pH '7. In the presence of excess sulfate ion,equilibrium is reached between the crystal and dissolved form; but oncethe crystals have been dissolved at higher pH, the addition of sulfuricacid or sulfates does not bring about crystallization. An aqueoussolution of the streptomycin sesquisulfate may be obtained by treating aslurry of the crystals in water with Ba (OI-I) 2 or with a syntheticresin acid-adsorbent or anion exchanger (e. g., Amberlite lit-4B, amodified phenol formaldehyde polyamine condensate) until neutral pH isobtained, warming the mixture, and filtering. Freeze-drying of thissolution yields streptomycin sesquisulfate having a potency (bio-assay)of about 860 units/mg. The product may be used per so as a therapeuticagent, or converted into pure dihydrostleptomycin (sesquisulfate) ingood yield.

Example 2 Replacement of the guanidine sulfate employed in Example 1with an equimolecular quantity of ammonium sulfate does not(qualitatively) affect the production of streptomycin sesquisulfate.

Example 3 Replacement of the 5% aqueous solution of guanidine sulfateemployed in Example 1 with half the volume of a 10% solution does not(qualitatively) affect the production of streptomycin sesquisulfate.

Example 4 The guanidine sulfate solution employed in Example 1 isreplaced with an equivalent amount of, a 5% aqueous solution ofguanidine phosphate; and methanol is: added to the separated aqueousphase until cloudiness occurs, and the solution allowed to stand. Thestreptomycin sesquiphosphate gradually crystallizing out is recovered byfiltration or centrifugation. It is soluble in water, insoluble inmethanol, and slightly soluble in 50% aqueous methanol. Its analysis (C,34.31%; H, 6.30%; N, 13.35%; and P, 6.58%) is in close agreement withthe theoretical.

Example 5 Replacement of the n-butanol employed in Example 1 by amylacetate, and addition of methanol to the separated aqueous phase untilcloudiness occurs does not (qualitatively) affect the production ofstreptomycin sesquisulfate; nor does addition of methanol tothen-butanol employed in Example 1 (e; g in a ratio of 1:5 by

volume).

Ezcample 6 nosidostreptomycin tergitate may be obtained,

for example, by dissolving 1.4 g. mannosidostreptomycintrihydrochloridein 75 ml. water, adding 7.5 ml. of a 25% solution'ofTergitol Penetrant 7 in water, while agitating; and, after the reactionmixture has stood for about an hour, filtering oif the precipitatedtergitate and drying it.

The preferred organic solvent for the practice of this invention in theproduction of streptomycin sesquisulfate is n-butanol, inasmuch as thissalt is quite insoluble'in water saturated with n-butanol. Y

The'preferred weak-base salts of sulfuric and phosphoric acids for thepurposes of this invention are the ammonium salts, especially the aminesalts, and notably the guanidine salts; the term ammonium beingemployedhereinin its generic sense, comprehendingorganically-substituted-ammonium, Among the other utilizable weak-basesalts of sulfuric'and phosphoric acids are aniline sulfate and pyridinesulfate. When a crude. streptomycin 'tergitate is employed, the organicsolvent solution thereof is preferably washed with very dilute aqueoussulfuric acid or phosphoric acid before treatment to convert thetergitate to the sulfate or phosphate respectively, e. g., with a lessthan 0.5% (preferably about 0.3%) sulfuric acid; this treatment removingimpurities and resulting in a lesscolored crystalline product.

The crystals obtained may lac-further purifiedby washing them-withaqueous methanol (of the order or 50-90%).

Preferably, the tcrgitate is used in substantially-dry form. Preferablyalso, the tergitate (especially the wet tergitate) is allowed to age fora period of the order of -25 days before treatment to obtain thecrystalline salts. Thus, aging of the wet streptomycin tergitate in acold room (under conditions preventing air-drying) for about 8 daysresulted in as high a yield of crystalline streptomycin sesquisulfate asobtained when using the dry tergitate, and approximately double theyield obtained when using the wet tergitate without aging. Preferablyalso, the tergitate is prepared by precipitation to a chemical assay endpoint, addition of excess Tergitol resulting in a lower yield of thecrystalline salts.

For optimum yields, there should be no delay in further processing afterdissolving the tergitate in the organic solvent.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

I claim:

1. The method of obtaining a crystalline salt of a streptomycin, whichcomprises: mixing (A) an organic-solvent solution of a salt-typecombination derived from the streptomycin and a member of the groupconsisting of the organic solvent being substantially waterimmiscibleand being a nonsolvent for the desired salt of streptomycin, and (B) anaqueous solution of a weak-base salt of a member of the group consistingof sulfuric acid and phosphoric acid; and recovering the formedcrystalline salt of the streptomycin.

2. The method of obtaining crystalline streptomycin sesquisulfate, whichcomprises: mixing (A) an organic-solvent solution of a salt-typecombination derived from streptomycin and.

the organic solvent being substantially waterimmiscible and being anonsolvent for streptomycin sesquisulfate, and (B) an aqueous solutionof a sulfuric acid salt of a weak base; and recovering the formedcrystalling streptomycin sesquisulfate.

3. The method defined by claim 2, in which the sulfuric acid salt of aweak base is an ammonium sulfate.

4. The method defined by claim 2, in which the sulfuric acid salt of aweak base is guanidine sulfate.

5. The method defined by claim 2, in which the organic solventessentially comprises n-butanol.

6. The method defined by claim 2, in which the organic solvent solutionis washed with very dilute aqueous sulfuric acid before treatment withthe sulfuric acid salt.

7. The method defined by claim 2, in which the salt-type combinationemployed is substantiallydry.

8. The method defined by claim 2, in which the salt-type combinationemployed is a wet material which has been aged for a period of the orderof 5 to 25 days.

LEON J. HEUSER.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 2,462,175 Folkers Feb. 22, 19492,531,869 Album et al Nov. 28, 1950 2,537,933 Lott et al. Jan. 9, 19512,537,934 Lott et a1. Jan. 9, 1951 2,537,941 Regna et a1 Jan. 9, 1951

1. THE METHOD OF OBTAINING A CRYSTALLINE SALT OF A STREPTOMYCIN, WHICHCOMPRISES: MIXING (A) AN ORGANIC-SOLVENT SOLUTION OF A SALT-TYPECOMBINATION DERIVED FROM THE STREPTOMYCIN AND A MEMBER OF THE GROUPCONSISTING OF